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Marie Yovanovitch, abruptly recalled as the US envoy to Ukraine in May this year, appeared for a closed-door deposition, according to Democratic lawmakers leading the inquiry, after she had been told by the State Department at the behest of the White House not to show up.
The lawmakers said they then issued a subpoena for her appearance and she complied.
Yovanovitch, according to a copy of her opening statement to lawmakers posted online by the Washington Post, said she was told by a senior State Department official about "a concerted campaign against me" and that Trump had pushed for her removal since the middle of last year even though the department believed "I had done nothing wrong."
She also expressed alarm over damage to American diplomacy under Trump and warned about "private interests" circumventing "professional diplomats for their own gain, not the public good."
The impeachment inquiry focuses on a July 25 phone call in which Trump pressed Ukrainian President Volodymyr Zelenskiy to investigate a domestic political rival, Democrat Joe Biden, and Biden's businessman son Hunter Biden.
Giuliani has accused Yovanovitch of blocking efforts to persuade Ukraine to investigate the Bidens. Giuliani has said he provided information to both Trump and the State Department about Yovanovitch, who he suggested was biased against Trump.
On Friday, Giuliani said: "I was doing it in my role as a defence lawyer" for Trump.
In her statement, Yovanovitch said she did not know Giuliani's motives for attacking her but that his associates "may well have believed that their personal financial ambitions were stymied by our anti-corruption policy in Ukraine."
Democrats have called her removal politically motivated.
"She's a brave woman," Democratic congressman Michael Quigley said of Yovanovitch during a break in the testimony.
According to a White House summary, Trump in his call to Zelenskiy described Yovanovitch in this way: "the woman was bad news and the people she was dealing with in the Ukraine were bad news."
Zelenskiy agreed with Trump that she was a "bad ambassador" and agreed to investigate the Bidens.
The conversation occurred after Trump had withheld $US391 million (US$616 million) in congressionally approved security assistance to Ukraine to help it deal with Russia-backed separatists in the eastern part of the country.
"Although I understand that I served at the pleasure of the president, I was nevertheless incredulous that the US government chose to remove an Ambassador based, as best as I can tell, on unfounded and false claims by people with clearly questionable motives," Yovanovitch said in her statement.
Yovanovitch entered the Capitol building for the deposition wearing dark glasses and walked past a crowd of journalists without responding to questions.
A career diplomat who also has served as US ambassador to two other countries, Yovanovitch's stint as envoy in Kiev was cut short when she was recalled to Washington as Trump allies leveled unsubstantiated charges of disloyalty and other allegations against her.
Democrats have accused Trump of pressuring a vulnerable foreign ally to dig up dirt on a domestic rival for his own political benefit. Biden, the former US vice president, is a leading Democratic contender for the right to face Trump in the November 2020 presidential election. Trump has denied he did anything wrong on the call.
The investigation of Trump could lead to the approval of articles of impeachment - or formal charges - against the president in the House. A trial on whether to remove him from office would then be held in the US Senate, where the Republicans who control the chamber have shown little appetite for ousting the president.
The chairmen of the three committees leading the impeachment inquiry warned the administration against stonewalling. Any efforts to prevent witness cooperation will be deemed obstruction of Congress "and an adverse inference may be drawn against the President on the underlying allegations of corruption and cover-up," they said in a statement.
Yovanovitch warned about Russia's "malign intentions" toward Ukraine and said that if the United States allows Russian actions toward its neighbor to stand "we will set a precedent that the United States will regret for decades to come."
"Today, we see the State Department attacked and hollowed out from within. State Department leadership, with Congress, needs to take action now to defend this great institution, and its thousands of loyal and effective employees," Yovanovitch said.
Yovanovitch said in her statement that the notion she had been disloyal to Trump was "fictitious" and dismissed the idea that former Democratic President Barack Obama's administration had asked her to harm Trump's 2016 presidential campaign.
Yovanovitch was the third key witness to appear in the impeachment inquiry. Gordon Sondland, the US ambassador to the European Union, will comply with a subpoena and testify next Thursday before the three House committees leading the impeachment inquiry, his lawyers said, despite the White House policy against cooperation.
But Sondland is not authorised to release the documents the committees have sought, his lawyers said, adding that he hopes the material will be shared with the committees before his appearance. Sondland was initially scheduled to testify before the House committees on Tuesday, but was blocked by the Trump administration from appearing.
On Thursday, two Florida businessmen - Ukraine-born Lev Parnas and Belarus-born Igor Fruman - who had helped Giuliani as he investigated Biden were arrested in what US prosecutors said was a scheme to illegally funnel money to a pro-Trump election committee and other political candidates.
Parnas sought the help of a US congressman to get Trump to remove Yovanovitch, according to the indictment.